Post-transcriptional Modifications of the tRNA Anticodon Stem and Loop (ASL) Affect the Ability of tRNA to Bind Synonymous Codons.

ABSTRACT

GUSTILO, ESTELLA MAILUM. Post-transcriptional Modifications of the tRNA Anticodon Stem and Loop (ASL) Affect the Ability of tRNA to Bind Synonymous Codons. (Under the direction of Dr. Paul F. Agris).

The Genetic Code is arranged into sixteen codon boxes, where the four codons in each box are similar in their first two letters but differ at the third position (the wobble position). In the universal Genetic Code, each amino acid, except for Tryptophan and Methionine that have one codon each, is encoded by two to six codons (two to six-fold degenerate). There are fewer tRNA species than codons; therefore, a tRNA species can recognize more than one codon. This flexibility in recognition resides in the third position of the codon:anticodon pairing, the wobble position.

anticodon restrict codon recognition to the two codons specific for that 2-fold degenerate amino acid. For example, Lysine has two codons (AAA and AAG) that share a codon box with Asparagine codons (AAU and AAC). The modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine at the wobble position of human tRNA (tRNALys mcm5s2U34) confers this tRNA’s ability to restrict codon recognition to the two Lysine codons only.

Post-transcriptional Modifications of the tRNA Anticodon Stem and Loop (ASL) Affect the Ability of tRNA to Bind Synonymous Codons

by

Estella M. Gustilo

A dissertation submitted to the Graduate Faculty of North Carolina State University

In partial fulfillment of the Requirements for the degree of

Doctor of Philosophy

Molecular and Structural Biochemistry

Raleigh, North Carolina June 5, 2009

APPROVED BY:

_______________________________ ______________________________

Dr. Paul F. Agris Dr. E. Stuart Maxwell

Committee Chair

DEDICATION

To my father who showed me the magic in books and the wonders of learning. To my

mother who expressed unconditional love. To my sister Marietta who offered unconditional

BIOGRAPHY

Estella M. Gustilo was born in the Philippines and immigrated to the United States with her

family at a young age. She grew up in Charleston, SC, where she attended college. Upon

graduation from college she worked in molecular biology research, where she found her

ACKNOWLEDGMENTS

The process of earning my Ph.D. has blessed me with wonderful opportunities of

learning. I will be forever grateful for those who contributed to my growth during these

years. First, I extend my deepest thanks to my mentor, Dr. Paul F. Agris, whose brilliance

and dedication to his craft has been inspiring. Dr. Agris has shown me amazing possibilities.

I thank each of the other members of my graduate committee; Dr. E. Stuart Maxwell, Dr.

Linda Spremulli, and Dr. Paul Wollenzien have given me valuable advice, collaborated in

these studies, and expanded my horizons.

I acknowledge my fellow lab members, especially William Darnell Graham, Dr.

Franck A. P. Vendeix, and William A. Cantara for providing me with dependable support.

My fellow biochemistry graduate students made this experience so much fun. Dr. Tatjana

Shapkina and Dr. Keith Gagnon have shared technical knowledge that has helped make this

study become more successful. Dr. Simpson Joseph has been extremely kind and opened his

lab and resources to me for several weeks to learn precious methods used in this work. This

study wouldn’t have been possible without technical training provided by Dr. Steven S.

Phelps. Steve has been such a wonderful, supportive friend who has taught me so much.

As always, my close family and friends have been instrumental in all the good things

that have come from my life. Even as science fills my mind, they fill my heart. My parents

and my sister Marietta have never failed me. Finally, but not least, I must thank the Creator,

whose mind I’ve seen only glimpses of in my study of science but whose heart I know fully.

TABLE OF CONTENTS

LIST OF TABLES………. vii

LIST OF FIGURES ……….. viii

CHAPTER 1. Post-transcriptional modifications are a trademark of tRNA…….….. 1

1.1 The roles of transfer ribonucleic Acid (tRNA) and the significance of post-transcriptional modifications to tRNA functions………..………. 1

1.2 The Genetic Code………..………. 7

1.3 The effects of post-transcriptional modifications of tRNA on synonymous-codon reading………..………….. 9

REFERENCES………..……… 16

CHAPTER 2. Modifications of the anticodon stem and loop of human tRNALys (ASLLysUUU) restrict codon recognition to Lysine codons AAA and AAG……...……... 24

2.1 Introduction………...……….. 24

2.2 Materials and Methods………...………. 26

2.3 Results………...……….. 28

2.4 Conclusions………...……….. 28

REFERENCES……….……..………... 32

CHAPTER 3. Anticodon domain modifications contribute order to tRNA for ribosome-mediated codon binding………...……….. 35

ABSTRACT………...………… 36

INTRODUCTION………. 39

EXPERIMENTAL PROCEDURE………..……..……… 42

RESULTS………..……… 47

DISCUSSION ……….….………. 62

CONCLUSION………..…..…….. 70

ACKNOWLEDGEMENTS……….……..…… 71

SUPPORTING INFORMATION AVAILABLE………..……..…….. 71

REFERENCES……….………. 72

ABSTRACT……….………….. 96

INTRODUCTION……….……… 96

MATERIALS AND METHODS……….………. 98

RESULTS AND DISCUSSIONS……….………….……... 107

FUNDING……….………… 114

ACKNOWLEDGEMENTS……….………. 114

REFERENCES……….………. 116

CHAPTER 5. The wobble position modified nucleoside of human mitochondrial tRNAMet decodes unconventional codons in the mitochondrial Genetic Code……….. 134

ABSTRACT……….. 135

INTRODUCTION……….……… 136

RESULTS……….………. 140

DISCUSSION ………... 146

MATERIALS AND METHODS……….………. 151

ACKNOWLEDGEMENTS……….…….………. 155

REFERENCES……….………. 157

CHAPTER 6. Summary and Insights……….……….. 172

6.1 The Evolution of tRNA and the Genetic Code……… 172

6.2 Implications of tRNA Modifications on Gene-Expression Regulation………... 175

6.3 tRNA codon-reading flexibility is Modulated by the Modifications at the Anticodon Loop……… 181

LIST OF TABLES

CHAPTER 1

Table 1. Distribution of modified nucleosides………. 11

Table 2. The recognition of tRNA wobble position-34 for the wobble position (N3) of the

codon………. 15

CHAPTER 3

Table 1. Thermal Parameters of Unmodified and Modified ASLVal3UAC ... 77

Table 2. Affinity of Unmodified and Modified ASLVal3UAC for the Valine Codons ……... 77

SUPPLEMENTARY MATERIALS:

Table 1. Local base step parameters of the unmodified ASL Val3UAC……….……….. 93

CHAPTER 4

Table 1. Thermodynamic contributions of f5C34………..……….. 125

CHAPTER 5

Table 1. Affinity of Unmodified and f5C34-Modified hmtASLMetCAU-Ψ27 for the

Mitochondrial Methionine Codons at E. coli Ribosomal P or A-site ………... 161

CHAPTER 6

Table 1. Contribution of modifications to codon recognition, frameshifting, and as

LIST OF FIGURES

CHAPTER 1

Figure 1. Bacterial tRNAs on the ribosome during translation……… 2

Figure 2. The tRNA……….………. 3

Figure 3. The Universal Genetic Code……….……… 8

CHAPTER 2

Figure 1. The anticodon stem and loop of human tRNALys3 (ASLLys3UUU-mcm5s2U34,

ms2t6A37)………..………….. 30

Figure 2. Ribosomal, equilibrium binding curves of the fully-modified human ASLLys3UUU-

mcm5s2U34, ms2t6A37 or the unmodified ASLLys3UUU………...…………. 31

CHAPTER 3

Figure 1. Nucleotide sequence of the Escherichia coli tRNAVal3UAC anticodon stem and loop

(ASLVal3UAC) and its modified nucleosides……… 82

Figure 2. UV-monitored, thermal transitions and circular dichroism specta of unmodified

and modified ASLVal3UAC……….……….. 83

Figure 3. Binding of unmodified and modified ASLVal3UAC to valine codons at the A-site of

E. coli 30S ribosomal subunits……….. 84

Figure 4. Detection of the base-paired imino protons of the ASLs by NMR………... 85

Figure 5. Superimposed 1H-1H COSY spectra of unmodified and modified ASLVal3UAC… 86

Figure 7. Solution structures of ASLVal3UAC -cmo5U34;m6A37 and ASLVal3UAC and the rmsd for each of their nucleotides……….……….. 88

Figure 8. Superimposition of the average structures of the loop residues of ASLVal3UAC -cmo5U34; m6A37 with that of ASLVal3UAC……….……….. 89

Figure 9. Loop structure of ASLVal3UAC-cmo5U34;m6A37 in solution compared to that of the crystallographic structure on the ribosome……… 90

SUPPLEMENTARY MATERIALS:

Figure 1. Superposition of the 1H-31P HETCOR spectra of ASLVal3UAC and ASLVal3

UAC-cmo5U34;m6A37………..…………. 91

Figure 2. Anomeric to aromatic connectivity for ASLVal3UAC and ASLVal3UAC-cmo5U34;m6A37 ……….……….. 92

CHAPTER 4

Figure 1. Human mitochondrial tRNAMetCAU ………... 126

Figure 2. Synthesis of the 5-formylcytidine phosphoramidite………... 127

Figure 3. NMR spectra of the hmASLMetCAU ………... 128

Figure 4. HPLC nucleoside composition and NMR analyses of hmASLMetCAU and f5C…... 129

Figure 5. Thermal denaturations and circular dichroism spectra of the hmASLMetCAU -Ψ27 and

the hmASLMetCAU -Ψ27;f5C34……….……. 130

Figure 6. Codon binding by hmASLMetCAU -Ψ27 and the hmASLMetCAU -Ψ27;f5C34……….. 131 Figure 7. Analysis of the pKa of cytidine in comparison to that of 5-formylcytidine……... 132

Supplemental Figure. Heavy atoms root mean square deviation (r.m.s.d.) variations as a function of the molecular dynamics simulation time for the hmASLMetCAU-Ψ27;f5C34 and for

the hmASLMetCAU-Ψ27 ………... 133

CHAPTER 5

Figure 2. Human mitochondrial tRNAMetCAU……….………….. 166

Figure 3. Ribosomal binding curves of the wobble-modified hmtASLMetCAU-Ψ27;f5C34 or the

hmtASLMetCAU-Ψ27……….……… 167

Figure 4. Binding kinetics of the hmtASLMetCAU-Ψ27;f5C34 and the hmtASLMetCAU-Ψ27 to AUG or AUA at the P- and A-sites……….……….. 168

Figure 5. Codon binding of the hmtASLMetCAU-Ψ27;f5C34 and the hmtASLMetCAU-Ψ27 on the bovine 55S mitochondrial ribosome……….. 169

Figure 6. Equilibrium binding of the hmtASLMetCAU-Ψ27;f5C34 or the hmtASLMetCAU-Ψ27 to the unconventional Methionine codons AUU, AUC, and AUA……… 170

Figure 7. The molecular dynamics simulation of the A:f5C base pair……….………. 171

CHAPTER 6

Figure 1. Accommodation of tRNA on the ribosome………... 174

Figure 2. Modifications of the anticodon stem and loop (ASL) of tRNA order the loop, prevent frameshifting and allow accurate codon selection……… 176

CHAPTER 1

Post-transcriptional modifications are a trademark of tRNA

1.1 The roles of transfer ribonucleic acid (tRNA) and the significance of

post-transcriptional modifications to tRNA functions.

Transfer ribonucleic acid (tRNA) is a crucial participant in protein biosynthesis. The

tRNA’s primary role occurs on the ribosome during translation of genetic information, where

the tRNA reads the codes for amino acids imbedded in messenger RNA (mRNA) and places

the appropriate amino acid into a growing protein (Figure 1). The Genetic Code is the

language of protein synthesis; it is stored in deoxyribonucleic acid (DNA), transcribed into

mRNA, and translated into proteins in the ribosome by the tRNA. Therefore, the tRNA is a

critical translator of the Genetic Code in gene expression. tRNA is the faithful keeper of the

Genetic Code, keeping fidelity from the start of protein synthesis with aminoacylation to

completion with the production of viable proteins. Inherent in its primary, secondary, and

tertiary structure is information that gives the tRNA its ability to function (Figure 2). The

tRNA’s function is dependent on its interactions with a variety of cellular molecules such as

aminoacyl-tRNA synthetases (aaRSs), translation initiation factors, translation elongation

factors, mRNAs, ribosomes, and peptidylhydrolases. Post-transcriptional modifications of

Figure 1. Bacterial tRNAs on the ribosome during translation. During initiation, the 30S

ribosomal subunit (transparent grey circle) binds the mRNA (light blue strip) and the initiator tRNA

at the ribosomal peptidyl (P)-site with the aid of initiation factors (not shown). The 50S subunit (dark

grey) then joins the complex to form the complete 70S ribosome. Amino acids are added to the

nascent protein during elongation, where an elongator aminoacyl-tRNA binds its codon on the mRNA

at the the aminoacyl (A)-site with the aid of elongation factors. A peptide bond is formed between

the amino acid bound to the tRNA at the P-site and the amino acid of the tRNA at the A-site, and the

entire protein chain is transferred to the tRNA at the A-site. The P-site tRNA then moves to the exit

(E)-site (not shown), and the tRNA with the growing protein at the A-site is translocated to the P-site.

Another elongator aminoacyl-tRNA binds its codon at the A-site until the termination stage, where

release factors terminate protein synthesis. Accuracy and efficiency are achieved when the

appropriate tRNAs bind their corresponding codon.

A

P

Tyr

Met

Lys

Ser

UCG AUG AGC UAC

Figure 2. The tRNA. A. The secondary cloverleaf structure of tRNA. B. The tertiary L-shaped

structure of tRNA. The anticodon triplet consists of positions 34, 35, and 36. The tRNA consists of

four main domains: the dihydrouridine stem and loop (DSL), the anticodon stem and loop (ASL), the

ribothymidine (T), pseudouridine (Ψ), cytidine (C) stem and loop (TSL). The tRNA also consists of a

segment of a variable number of nucleosides (VL). Positions 34 and 37 are often

post-transcriptionally modified.

TSL

ASL

DSL

AAS

5

’

3

’

ASL

DSL

TSL

AAS

VL

3

’

5

’

34

37

34

37

Approximately 100 different modified nucleosides have been found in RNA [5].

tRNA is the most highly modified nucleic acid in the cell and consists of the greatest variety

of modification chemistries [6]. Post-transcriptional modifications decorate nucleosides all

along the tRNA and are integral to its purpose [1-4]. Up to thirty percent of tRNA

nucleosides at more than sixty different positions are modified [6]. Modified nucleosides are

derivatives of the four ribonucleic acid monomers Adenosine (A), Guanosine (G), Uridine

(U), and Cytidine (C), and therefore add additional chemistries to the limited number of only

four main nucleosides. The significance of modified nucleosides in the tRNA is evident in

their conservation among species and the amount of genetic material assigned to their

synthesis. In bacteria, approximately one percent of the genome codes for tRNA modifying

enzymes - this is four times more than the approximately 0.25 percent of bacterial genes

designated to tRNA [7]. Therefore, more genetic information is allocated to the modifying of

tRNA than to the tRNA themselves [7]. Modifications help the tRNA fold into its secondary

cloverleaf structure and tertiary L-shaped conformation mandatory for function and may aid

in the tRNA’s many interactions with proteins [2, 8-12]. Modifications are important to the

stability of tRNA and thus, affect its half-life [13-14].

Aminoacylation of the tRNA is the first step of protein synthesis. Aminoacyl-tRNA

synthetase charges the tRNA with the correct amino acid. Fidelity of translation begins at

aminoacylation. Modifications on the tRNA are specific identity determinants by some

aminoacyl-tRNA synthetases and therefore important to the aminoacylation of these tRNAs

[15-19]. Modifications may also affect aminoacylation via the modifications’ contributions

unmodified) can be aminoacylated, there are small differences in kinetics, suggesting that

modified nucleosides can be of some influence [11]. This is likely due to the inability of

tRNA to fold into its functional conformation. Unmodified tRNAs also have a greater

probability of being mischarged by a noncognate aminoacyl-tRNA synthetase [21, 22].

In the succeeding steps to the production of functional proteins, tRNA interacts with

many proteins such as inititation factors or elongation factors, and the proteins of the

ribosome. In the ribosome, it decodes the sequence of mRNAs into sequences of protein.

The tRNA is not a passive adaptor in translation but an active player during its function by

undergoing conformational changes [23-25]. Being the linker between genetic language and

end product (protein), the tRNA’s accuracy is necessary if the cell is to produce functional

proteins. And this fidelity must be balanced with the high speed of translation (over 20

peptide bonds per second). Post-transcriptional modifications at or near the anticodon of

tRNA are often required for accurate and efficient translation of the codons of mRNA [1, 11,

25-35].

The tRNA is an ancient molecule that is a remnant of the RNA World, where cellular

processes were carried out solely by RNA [36]. Thus, the tRNA has evolved into a

multifunctional molecule in the cell and is involved in a variety of processes in the cell.

While its main job is the adaptor molecule in protein synthesis, the tRNA has been found to

have other roles outside of translation on the ribosome [37]. The tRNA has been found to

function in viral proliferation [38-40], gene expression regulation [41, 42], cell division/DNA

chlorophyll synthesis [50]. Post-transcriptional modifications of tRNA may be directly or

indirectly involved in the functions of tRNA [11, 51].

The tRNA has been shown to be an important player in retroviral replication [38-40].

A specific tRNA isoacceptor, tRNALys3, is recruited by infected cells as the primer for the

reverse transcription of human immunodeficiency virus (HIV-1) [39, 52-55]. This has placed

specific tRNAs in study as potential drug targets [38]. The tRNALys3 consists of specific

modifications at the anticodon loop [30]. These modifications play a role in HIV-1’s

specificity for tRNALys3 and stabilize the tRNA Lys3’s interaction with the HIV-1 genome

[56-58].

The tRNA is also involved in DNA replication and/or cell division. Specific Serine

and Arginine tRNA isoacceptors have been shown to affect the cell cycle [59, 60]. The

modifications of tRNA may be involved in cell cycle regulation in that certain tRNA

isoacceptors with specific modifications are apparent in certain cell cycle stages [61-63].

The degree of tRNA modifications’ importance in many cellular processes has yet to

be fully appreciated. tRNA modifications are most studied in the context of tRNA’s central

role of translation. The modifications at tRNA’s anticodon stem and loop (ASL) are

especially important, as they have been shown to be significant factors in codon: anticodon

pairing, reading frame maintenance, translocation of tRNA from the ribosomal aminoacyl

(A)-site to the peptidyl (P)-site, and the balance between decoding accuracy and speed

[64-66]. The tRNA position 34 (wobble position) and position 37 at the ASL are often modified

Modifications at these two specific positions affect tRNA’s main function of reading codons

on the ribosome.

1.2 The Genetic Code

Proteins are produced by the cell using instructions compiled in the Genetic Code

(Figure 3). The Genetic Code is stored in DNA, transcribed into mRNA, and translated on

the ribosome by tRNA. The elucidation of the Genetic Code is arguably the greatest

accomplishment in the biomedical research of the 1960s. Upon its discovery, the Genetic

Code was considered universal in that there appeared to be little variation among organisms.

The Genetic Code consists of 64 codons: 61 sense codons that code for any of the 20 amino

acids and three termination codons. In the universal Genetic Code, most amino acids have

two to six codons. The different codons that code for the same amino acid are synonymous

codons. Only Tryptophan and Methionine have no synonymous codons, as these two amino

acids only have one codon each.

The Genetic Code is arranged into sixteen sets of four codons, portrayed in a table of

sixteen codon boxes (Figure 3). The four codes of a codon box are similar to each other in

the first two letters and differ only in the third letter. Some amino acids are encoded in

synonymous codons all residing in the same codon box. Other amino acids share the codes

of a codon box; these codons are termed to be in a “split box.” For example, Valine is

have two codons that share a codon box. Thus, the codons of Asparagine and Lysine are

very similar and differ only in the third position, the wobble position of the codon.

Figure 3. The universal Genetic Code. There are 61 sense codons that code for the 20

amino acids and 3 stop codons. The Genetic Code is arraigned into 16 codon boxes. Each

codon box consists of four codons that have the same first two letters. The four codons of a

box differ only in their third letter (the wobble position).

UUU UUC UUA UUG Phe Phe Leu UCU UCC UCA UCG Ser Ser Ser Ser UAU UAC UAA UAG Tyr Tyr Stop Stop UGU UGC UGG Cys Cys Trp CUU CUC CUA CUG Leu Leu Leu Leu CCU CCC CCA CCG Pro Pro Pro Pro CAU CAC CAA CAG His His Gln Gln CGU CGC CGA CGG Arg Arg Arg Arg AUU AUC AUA AUG Ile Ile Ile Met ACU ACC ACA ACG Thr Thr Thr Thr AAU AAC AAA AAG Asn Asn Lys Lys AGU AGC AGA AGG GUU GUC GUA GUG Val Val Val Val GCU GCC GCA GCG Ala Ala Ala Ala GAU GAC GAA GAG Asp Asp Glu Glu GGC GGA GGG Gly Gly Gly

U

C

A

G

The Genetic Code was once thought to be “frozen” in that it was considered universal

among all protein-producing systems [67]. However, the sequencing of the human and

bovine mitochondrial DNA in 1981 and 1982, respectively, has proven that the Genetic Code

can deviate from the Code established from Escherichia coli in the 1960s [68, 69]. Soon

after the sequencing of the mammalian mitochondrial DNA, other deviations from the

universal Genetic Code were found [70-72].

In the ribosome, the tRNA must decipher the Genetic Code with great accuracy and

efficiency. tRNA modifications at the anticodon loop have been shown to facilitate accurate

and efficient reading of codons. ASL modifications aid in the recognition of synonymous

codons by restricting codon recognition of split boxes or expanding codon recognition of

whole boxes [1]. Certain ASL modifications also allow the reading of unconventional

codons in the alteration of the universal Genetic Code [73]. Thus, modifications at the ASL

modulate the tRNA’s decoding capacity.

1.3 The effects of tRNA post-transcriptional modifications on synonymous-codon

recognition

The Genetic Code is redundant or degenerate in that there are 61 codons for only 20

amino acids. Codons also outnumber the tRNA species that decode them. In E. coli, there

are 45 tRNA species [74] that must translate the 61 codes; therefore, on average, there are

only some three tRNAs for four codons. Francis Crick’s Wobble Hypothesis explained that a

position (the wobble position) of codon:anticodon binding [75]. Crick hypothesized that the

first two positions of codon:anticodon pairings followed Watson-Crick nucleoside base

pairing rules of Adenosine (A) pairing with Uridine (U) and Guanosine (G) pairing with

Cytosine (C). However, the third position (wobble position) is more flexible in that a G can

pair with its cognate C or “wobble” to a U. Also, Crick explained that Inosine (I), a modified

A, can exhibit a great deal of flexibility by pairing with U, C, or A.

The forty-plus years of research since Crick’s hypothesis has revealed that, although

Crick was fundamentally correct in his assumption of pairing flexibility,

wobble-pairing can be more complicated than just G:U and Inosine wobble-pairings. Oftentimes, the wobble

position of tRNA (position 34, first position of the anticodon) is modified, and these

modifications are significant to tRNA’s ability to wobble to a particular codon (Table 1) [76].

tRNA position 37 is also often modified and aid in codon recognition (Table 1).

Modified nucleosides at the anticodon stem and loop (ASL) of tRNA often give the

tRNA its ability to recognize synonymous codons. Some modifications at the ASL restrict

codon recognition to a few synonymous codons while other modifications serve to extend

codon recognition to include synonymous codons (Table 2). The ability of ASL

modifications to restrict or expand codon reading is due to the extent of degeneracy of the

codons. For example, Lysine (Lys) is two-fold degenerate in that it has two codons, AAA

and AAG. The two Lys codons share a codon box with Asparagine (Asn). There are two

Asn codons – AAU and AAC. Thus, Lys and Asn codons are similar, with the only

The human tRNALys3UUU is modified with 5-methoxycarbonylmethyl-2-thiouridine at

position 34 (wobble position of tRNA) and 2-methylthio-N6-threonylcarbamoyladenosine at

position 37 (tRNALys3UUU-mcm5s2U34, ms2t6A37). Chapter 2 describes how the specific

modifications at the ASL of tRNALys3UUU (ASLLys3UUU-mcm5s2U34, ms2t6A37) serve to limit

codon recognition to only the two Lys codons, AAA and AAG.

On the other hand, Valine (Val) is encoded by four codons. Thus, Val is four-fold

degenerate. All four Val codons reside in the same codon box; therefore, Val codons are

unique from the codons of the other amino acids. Chapter 3 explains how a 5-oxyacetic acid

modification at the wobble Uridine and N6-methyladenosine at position 37 of the anticodon

stem and loop of tRNAValUAC (ASLValUAC-cmo5U34, m6A37) are not required to bind the

tRNA’s cognate codon GUA but required to read the other Val codons of the GUN codon

box. Thus, the modifications of ASLValUAC-cmo5U34, m6A37 allow expansion of codon

recognition. The tRNAValUAC-cmo5U34, m6A37 is an example of one ASL-modified tRNA

that can read the synonymous codons from the same codon box.

tRNA modifications can also serve to expand codon recognition to include

unconventional codons. The Genetic Code varies to a certain extent among certain

organisms and semiautomous organelles. Various codon reassignments can be found in the

translational systems of the semiautomous organelles, mitochondria and chloroplasts, of

different organisms. The mammalian mitochondrial Genetic Code deviates from the

universal Genetic Code in that the universal termination codon UGA codes for Tryptophan;

the two universal Arginine codons AGA and AGG are stop codons, and the universal

In the universal Genetic Code, AUG is the start codon and the sole codon for

Methionine. Genetic translation occurs in three phases: initiation, elongation, and

termination. Initiation is when the mRNA binds the small ribosomal subunit and the initiator

tRNA reads the start codon at the ribosome’s peptidyl (P)-site. Initiation is aided by

initiation factors. After initiation, elongation causes the growth of the polypeptide chain until

completion with the termination step. In the cytoplasm, two tRNA species recognize AUG in

accordance to the codon’s placement on the ribosome. At initiation, AUG is at the P-site of

the ribosome and is recognized by the initiator tRNAMetCAU with the aid of initiation factors.

During elongation, elongator tRNAMetCAU reads AUG at the aminoacyl (A)- site of the

ribosome, facilitated by elongation factors. Mammalian mitochondrial translation differs

from cytoplasmic translation in that 1) there are two Met codons AUG and AUA, 2) only one

tRNAMetCAU recognizes both codons AUG and AUA, and 3) this one tRNAMetCAU acts in both

initiation and elongation. Interestingly, this one mitochondrial tRNAMetCAU is modified at the

wobble position with a 5-formyl group (mtRNAMetCAU-f5C34) [73, 78, 79]. Cytoplasmic

initiator tRNAMetCAU is unmodified at the wobble position. Cytoplasmic elongator

tRNAMet

CAU is modified at the wobble position with a 2’O-methyl group at the ribose moiety. Bacterial initiator tRNAMetCAU is unmodified at the wobble position, while the bacterial

elongator tRNAMetCAU is wobble-modified with N4-acetylcytidine (tRNAMetCAU-ac4C34).

Chapter 4 reports the first chemical synthesis of modified human mitochondrial ASLMet

(hmtASLMetCAU-f5C34) and the significance of the 5-formyl modification in reading the

For some mRNAs in the mitochondria, the universal Isoleucine codons AUU and

AUC are used as initiation codons. Chapter 5 shows how the 5-formyl group enhances

binding to the unconventional Met codon AUA at the ribosomal P- and A-sites and expands

codon recognition to include the unconventional start codons AUU and AUC at the P-site.

Thus, the 5-formyl modification of hmtRNAMetCAU-f5C34 allows codon-reading extension to

include the entire codon box AUN. Furthermore, these results demonstrate how a

modification at the ASL enhances the reading of unconventional codons in the reassignment

Table 2. The recognition of tRNA wobble position-34 for the wobble position (N3) of

the codon. xm5U includes all 5-methylene uridine derivatives, excluding 2-thio; s2U

includes all 2-thiouridine derivatives, and xo5U consists of all 5-oxyuridine derivatives.

a _f5_{C refers to the wobble-modified Cytidine found in mitochondrial tRNA}Met_.

b _{Recognition of codon N3 at the P-site.}

Anticodon N34 Codon N3

G C, U

C G

I U, C, A

xm5U G

s2_{U A, G}

xo5U A, G, U, C

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mitochondrial transfer RNAMet to decode AUG and AUA codons. Biochimie, 77, 104–108.

CHAPTER 2

Modifications of the anticodon stem and loop of human tRNALys (ASLLysUUU) restrict

codon recognition to Lysine codons AAA and AAG.

2.1 Introduction

The universal Genetic Code consists of 64 codons arranged into sixteen codon boxes.

Each codon box is composed of four codons that are similar in their first two letters and

differ only in their third letter. In the ribosome, this third letter of the codon binds to the first

letter of the tRNA anticodon to form the wobble pair. The two modified nucleosides

5-methylaminomethyl-2-thiouridine or a derivative at the wobble position-34 (mnm5s2U34) and

N6-threonylcarbamoyladenosine or a derivative at position 37 (t6A37) are found in almost all

tRNAs that read A or G in the wobble (third) letter of Glutamine (Gln), Lysine (Lys), and

Glutamate (Glu) codons [1-5]. Modifications at the wobble position and position 37 of

tRNAGln, tRNALys and tRNAGlu are also required for efficient aminoacylation by the tRNAs’

cognate synthetases [6-10]. In bacteria, these two modifications within the anticodon loop of

tRNALys (ASLLysUUU- mnm5s2U34, t6A37 or derivatives) have been found to be crucial to the

structure of the anticodon loop, and therefore credited to the tRNA’s ability to perform its

function on the ribosome [4]. The 5-methylaminomethyl modification of tRNALys’s wobble

position 34 (tRNALysUUU- mnm5U34) has been implicated in the recognition of the Lys codons

AAA and AAG, the maintenance of the reading frame, and translocation from the aminoacyl

significantly to the stability of the U:A base pairs, whereas the mnm5-group enhances

recognition of G. E. coli has but one gene for tRNALys and it has the mnm5s2U34UUt6A37

anticodon loop modification. The codon preference is for AAA.

The human tRNALys3 consists of the modifications

5-methoxycarbonylmethyl-2-thiouridine at the wobble position and 2-methylthio-N6-threonylcarbamoyladenosine at

position 37 (tRNALys3UUU-mcm5s2U34, ms2t6A37). Humans have two other Lys tRNAs,

isoacceptors 1 and 2. The anticodon sequence and modifications for tRNALys1, 2 are

C34UUt6A37, which decode the codon AAG. Codon preference in human cells is for AAG.

However, tRNALys3has come under scrutiny for its ability to act as the primer for reverse

transcription of HIV-1 and HIV-2 genomes [13-16]. The details to why tRNALys3UUU

-mcm5s2U34, ms2t6A37 is specifically chosen by the virus are not entirely clear. The

modifications of tRNALys3UUU-mcm5s2U34 are important in HIV’s ability to utilize tRNA Lys3

for annealing to the viral genome and for reverse transcription [17-18].

Derivatives of the 5-methylaminomethyl-2-thio modification at the wobble position

(xm5s2U34) and N6-threonylcabamoyladenosine (xt6U37) at position 37 of tRNA are highly

conserved among organisms of Archea, Bacteria, and Eukarya [1]. Most of our current

understanding of the functions of tRNA modifications is a result of original studies with

bacterial translational systems. Far fewer studies have focused on the effects of

modifications on the functions of tRNAs of higher organisms. Recently, the

5-methoxycarbonylmethyl-2-thiouridine at the wobble position of tRNALys3 (tRNALys3UUU

-mcm5_s2_U

ms2t6A37 (ASL Lys3UUU-mcm5s2U34, ms2t6A37; Figure 1). As in bacteria, the modifications at

the anticodon loop of tRNA restrict codon binding to the Lys codons AAA and AAG. This

function prevents of binding of the tRNALys3 to the similar Asn codons in the split codon

box.

2.2 Materials and Methods

Ribosomal Binding Assay

The ribosomal binding assays consisted of reaction mixtures of purified E. coli (MRE

600) 70S ribosomes, chemically synthesized mRNAs, and chemically synthesized ASLs.

The primary sequences of the 27-nt mRNA oligo nucleotides were derived from T4 gp32

mRNA [19] and were purchased from Dharmacon (ThermoFisher, Lafayette, CO). In order

to study binding at the ribosomal A-site, the P-site needs to be blocked or occupied.

Therefore, we designed the mRNA with the Methionine (Met) codon AUG at the P-site. The

E. coli tRNAfMet was then used to saturate the P-site. The mRNA sequences were tested for a

low probability of any secondary structure using the program RNA Structure 4.2 [20]. The

mRNA sequences synthesized for our studies were as follows (Lys codons AAA and AAG

are in bold):

1) 5’-GGCAAGGAGGUAAAAAUGAAAGCACGU-3’;

2) 5’-GGCAAGGAGGUAAAAAUGAAGGCACGU-3’.

The 70S ribosomal subunits were isolated as previously described [21]. The ASLs were

5’-end-labeled using [γ-32_{P] ATP (MP Biomedicals). Unlabeled ASLs in a range of}

detectable (2,000-5,000 CPM) of 5’-end, 32P-labeled ASLs in a fixed ratio of unlabeled ASL

to labeled ASL, in order to maintain radiochemical-specific activity. The assay was

performed in ribosomal binding buffer [50 mM HEPES, pH 7.0; 30 mM KCl; 70 mM

NH4Cl; 1 mM DTT; 100 μM EDTA; 20 mM MgCl2). Ribosomes (0.25 μM) were activated

by heating to 42 °C, incubating for 10 minutes and then slowly cooled to 37 °C. The

ribosomes were programmed with 2.5 μM mRNA for 15 minutes at 37 °C. To experiment

with the A-site, the P-site was saturated with E. coli tRNAfMet (Sigma-Aldrich) for 15

minutes at 37 °C. tRNAfMet binds to the Met codon AUG; see underlined codons of the

mRNA sequences above. Binding of ASLLys to the A-site was allowed to proceed for 30 min

at 37 °C. The reaction mixtures (20μL each) were then placed on ice for 20 minutes, diluted

with 100 μL buffer per reaction mixture, and filtered through nitrocellulose in a modified

Whatman Schleicher and Schuell (Brentford, U.K.) 96-well filtration apparatus [22]. Prior to

filtration of experimental samples, the nitrocellulose filter was equilibrated in binding buffer

at 4 °C for at least 20 min and each well of the filtration apparatus was washed with 100 μL

of cold binding buffer. After filtration of reaction samples, each well was then washed twice

with 100 μL of cold ribosomal binding buffer. The nitrocellulose was blotted dry with kim

wipes, and the radioactivity was measured using a phosphorimager (Molecular Dynamics,

GE Healthcare). Data were measured for radioactive intensity using the program ImageQuant

(Amersham). Nonspecific binding was determined by the binding of ASLs to ribosomes

without mRNA and subtracted from the experimental data. The final data is a result of at

least two separate experiments, each done with samples in triplicate, i.e. at least six results

2.3 Results

We have previously assessed the significance of modifications at the anticodon stem

and loop of the bacterial tRNALys (ASLLysUUU-mnm5U34, t6A37) in binding the Lys codons

AAA and AAG [4-5]. While either modification at the wobble position or position 37

enhanced codon binding over the unmodified ASLLys approximately seven-fold, the

modifications in combination increased binding by over ten-fold that of the unmodified

ASLLys. Our experiments with the anticodon stem and loop of human tRNALys3 (ASLLys3UUU

-mcm5s2U34, ms2t6A37) revealed similar results. In comparison to the modified ASLLys3UUU

-mcm5s2U34, t6A37 in in vitro ribosomal binding assays, the unmodified ASLLys3UUU showed

very poor binding to the Lys codons AAA and AAG (Figure 2). The binding of the

unmodified ASLLys to either Lys codon AAA or AAG was nearly undetectable unless

modifications at positions 34 and 37 were present (Figure 2). The modified ASLLys3UUU

-mcm5s2U34, ms2t6A37 bound AAA with a Kd of 3.1 ± 0.4 μM and AAG with a Kd of 3.9 ± 0.8

μM.

2.4 Conclusions

tRNA modifications are highly conserved among all three kingdoms. In split boxes,

the difference between codons of two different amino acids resides in the wobble position

(third letter) of the codon. Because codons in the same box are very similar, recognition of

split boxes must be stringent. tRNAs that decode A or G at the wobble position of split

boxes tend to have the xm5_s2_U

t6A37. In humans, the tRNA’s ASL is modified with derivatives of the bacterial

modifications: mcm5s2U34 and ms2t6A37. These modifications are been found to be highly

significant to this tRNA’s ability to decode the Lys codons AAA and AAG. Here we report

that the modifications at the anticodon stem and loop of human tRNALys3 (ASLLys3UUU

-mcm5s2U34, ms2t6A37) are required for the anticodon to bind to the Lys codons AAA and

AAG. According to Crick’s Wobble Hypothesis, the unmodified ASLLys3UUU should bind

AAA and AAG because the anticodon UUU is a cognate or Watson-Crick (W-C) pair for

codon AAA and the wobble pair for codon AAG [23]. However, we found that the

unmodified ASLLys showed very low levels of binding in in vitro ribosomal binding assay.

Only with the addition of the naturally-occurring modifications

5-methoxycarbonylmethyl-2-thiouridine to the wobble position and 2-methylthio-N6-threonylcarbamoyladenosine to

position 37 (ASLLys3UUU-mcm5s2U34, ms2t6A37), does the ASL bind both codons AAA and

AAG. Considering their significance to tRNALys3’s central role of decoding, modifications at

the ASL of human tRNALys3 may be have implications on other functions of the tRNALys,

such as its involvement in retroviral replication.

In addition to the quantitative results of codon binding reported here we now have the

NMR solution structure of the ASLLys3 and the X-ray crystallographic structures of the

ASLLys3UUU-mcm5s2U34, ms2t6A37 bound to both Lys codons AAA and AAG. A comparison

of the solution structure to that of the structure of the ribosomal X-ray indicates that the

modifications have restricted motional dynamics that directs the structure toward that

required for codon binding. These results are comparable to structures of bacterial ASLLys

Figure 1. The anticodon stem and loop of human tRNALys3 (ASLLys3UUU-mcm5s2U34,

ms2_t6_A

37). The ASL is modified at the wobble position-34 with

5-methoxycarbonylmethyl-2-thiouridine, at position 37 with 2-methylthio-N6-threonylcarbamoyladenosine, and at

position 39 with a pseudouridine.

U

Ψ A

C G A U G C A Ψ

A C

U

U U C G A G

A Ψ39

C

U ms_ms22_tt66A₃₇

mcm 5_s2_U

34 U U

G C

Pseudouridine,Ψ

5‐methoxycarbonylymethyl‐2‐thiouridine, 

mcm5_s2_U

2‐methylthio‐N6‐threonylcarbamoyladenosine, ms2_t6_A A A A

G A A 5’

5’ 3’

Figure 2. Ribosomal, equilibrium binding curves of the fully-modified human

ASLLys3UUU- mcm5s2U34, ms2t6A37 (▲) or the unmodified ASLLys3UUU (■). E. coli 70S

ribosomes were programmed with the Lysine codon A. AAA or B. AAG at the A-site. The

P-site was saturated with E. coli tRNAfMet, which binds to its cognate Methionine codon

AUG.

0

1

2

0

1

2

0

25

50

75

100

Bindi

n

g (

%

)

ASL

(

μ

M)

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Chapter 3

Anticodon Domain Modifications Contribute Order to tRNA for Ribosome-Mediated Codon Binding

Franck A. P. Vendeix, Agnieszka Dziergowska, Estella M. Gustilo, William D. Graham, Brian Sproat, Andrzej Malkiewicz, and Paul F. Agris.

Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, North Carolina 27695-7622, Institute of Organic Chemistry, Technical University, Zeromskiego 116, 90-924 Lŏdű, Poland, and Integrated DNA Technologies BVBA, Provisorium 2, Minderbroedersstraat 17-19, B-3000 Leuven, Belgium

Anticodon Domain Modifications Contribute Order to tRNA for

Ribosome-Mediated Codon Binding†,‡

Franck A. P. Vendeix‡, Agnieszka Dziergowska§, Estella M. Gustilo‡, William D. Graham‡,

Brian Sproat║_{, Andrzej Malkiewicz}§_{, and Paul F. Agris*},‡

Department of Molecular and Structural Biochemistry, North Carolina State University, 128

Polk Hall, Raleigh, North Carolina, USA 27695-7622, Institute of Organic Chemistry,

Technical University, Żeromskiego 116, 90-924, Łódź, Poland, and ║Integrated DNA

Technologies BVBA, Provisorium 2, Minderbroedersstraat 17-19, B-3000 Leuven, Belgium

Running Title: Wobble Modifications Order tRNA's Anticodon

†_{This work was supported by grants from the National Institutes of Health (Grant}

2-RO1-GM23037 to PFA), the National Science Foundation (MCB-0548602 and 2-RO1-2-RO1-GM23037

to PFA), RNA-TEC NV, and the Polish Ministry of Science and Education (to AM).

*_{To whom correspondence should be addressed.}

Phone: 1-919-515-6188.

E.Mail: Paul_Agris@ncsu.edu

FAX: 1-919-515-2047.

‡_{North Carolina State University}

║_{Integrated DNA Technologies}

1_{Abbreviations: ASL, anticodon stem and loop domain; ASL}Val3

UAC, ASL of E. coli's valine tRNA isoaccepting species 3 with anticodon UAC; CD, circular dichroism

spectropolarimetry; cmo5U34, uridine-5-oxyacetic acid; HPLC, high performance liquid

chromatography; m6A37, N6-methyladenosine-37; NOE, nuclear Overhauser effect; Tm,

ABSTRACT

The accuracy and efficiency with which tRNA decodes genomic information into

proteins require posttranscriptional modifications in or adjacent to the anticodon. The

modification uridine-5-oxyacetic acid (cmo5U34) is found at wobble position 34 in a single

isoaccepting tRNA species for six amino acids, alanine, leucine, proline, serine, threonine,

and valine, each having 4-fold degenerate codons. cmo5U34 makes possible the decoding of

24 codons by just six tRNAs. The contributions of this important modification to the

structures and codon binding affinities of the unmodified and fully modified anticodon stem

and loop domains of tRNAVal3UAC (ASLVal3UAC) were elucidated. The stems of the

unmodified ASLVal3UAC and that with cmo5U34 and N6-methyladenosine, m6A37, adopted an

A-form RNA conformation (rmsd 0.6 Å) as determined with NMR spectroscopy and

torsion-angle molecular dynamics. However, the UV hyperchromicity, circular dichroism

ellipticity, and structural analyses indicated that the anticodon modifications enhanced order

in the loop. ASLVal3UAC-cmo5U34;m6A37 exhibited high affinities for its cognate and wobble

codons GUA and GUG, and for GUU in the A-site of the programmed 30S ribosomal

subunit,whereas the unmodified ASLVal3UAC bound less strongly to GUA and not at all to

GUG and GUU. Together with recent crystal structures of ASLVal3UAC-cmo5U34;m6A37 bound

to all four of the valine codons in the A-site of the ribosome’s 30S subunit, these results

clearly demonstrate that the xo5U34 -type modifications order the anticodon loop prior to

A-site codon binding for an expanded codon reading, possibly reducing an entropic energy

INTRODUCTION

Transfer RNA is one of the most understood biological macromolecules. The

relationship of its nucleoside chemistry and oligonucleotide structure to its functions in

protein synthesis has been studied extensively (1, 2). Those studies have led to the

identification of more than 70 different posttranscriptional modifications present in tRNAs

(3). tRNA modifications increase stability (4), enhance decoding (5), restore ribosomal

binding (6), and influence reading frame maintenance (7, 8). In the course of translation,

anticodon domain modifications in particular play important roles in the accuracy and

efficiency of protein synthesis. Therefore, the modifications that occur at anticodon wobble

position 34, and at the conserved purine 37, 3′-adjacent to the anticodon, are of obvious

interest in tRNA’s decoding of mRNA (Figure 1). These modifications play critical and

distinctive roles in tRNA’s accurate and efficient binding of cognate and wobble codons

within the ribosome’s A-site (9).

Some 40 years ago, Francis Crick explained how a limited number of tRNAs could

decode the 61 amino acid codons (10). Our view of codon recognition by tRNA was then

altered in the modified wobble hypothesis to accommodate new information about

modifications (11). The limited number of tRNAs requires most tRNAs to read more than

one codon. Some tRNAs respond to codons in “mixed” codon boxes where distinction of the

third codon base (the most degenerate of coding positions) is important for discriminating

between the correct cognate or wobble codons and the incorrect but near-cognate codons. For

example, a wobble position, 2-thiouridine 34 (s2_U

12). In contrast, other modification chemistries expand wobble codon recognition, such as

tRNA’s wobble position inosine 34 (I34) that will bind codons ending in A3, C3, or U3 (12).

Modifications expand tRNA’s codon recognition beyond that envisioned in the wobble

hypothesis. Crick suggested that a U34 would pair with an A and wobble to a G in the third

position of the codon (A3 or G3) but would not base pair with a U3 or C3. He argued that a

U34 paired with a U3 or C3 would markedly distort the anticodon-codon minihelix (10).

However, wobble position U34 of some tRNA species will base pair with a U3 and even a

C3 in recognizing all of the synonymous codons of a 4-fold degenerate codon box (9, 13–

16). For tRNAs to recognize codons ending with a U3 or C3, as well as codons ending with

A3 and G3, modification of wobble position U34 is essential (9, 13–16). The modified

nucleoside capable of binding to A, G, U, and C is uridine-5-oxyacetic acid, cmo5U341

(Figure 1). Each of the six amino acids, alanine, leucine, proline, serine, threonine, and

valine, having 4-fold degenerate codons, has a single isoaccepting tRNA species containing

the modification cmo5U34 (17), but would a tRNA species with cmo5U34 be sufficient for cell

viability in the absence of all other isoaccepting tRNAs in vivo? The function of cmo5U34 in

tRNAPro _{was analyzed by introducing null copies of the two genes (cmoA and cmoB)}

identified as part of the cmo5U34 synthetic pathway into a strain of Salmonella having only

one of three tRNAPro isoacceptors, that with the wobble cmo5U34 modification (18). Growth

of this and other mutant strains demonstrated that all four proline codons (CCU/C/A/G) were

read by the cmo5U-containing tRNAPro, and that the complete modification was critical for

reading codons ending in U and C (18). However, the physicochemical contributions of